Field of the Invention
The present invention relates to a method for detecting a target nucleic acid molecule using a photocrosslinking reaction.
The present application claims priority on the basis of Japanese Patent Application No. 2012-063682, filed in Japan on Mar. 21, 2012, the contents of which are incorporated herein by reference.
Description of the Related Art
Nucleic acid molecules in a sample solution can be detected by a hybridization method using a labeled probe that specifically hybridizes with the nucleic acid molecule. As an example of a method thereof, after hybridizing a labeling probe preliminarily immobilized on beads with a target nucleic acid molecule, the nucleic acid molecule that has been hybridized and immobilized on the beads is precipitated on the bottom of a container. The temperature of the reaction liquid is subsequently gradually raised to a temperature equal to or higher than the denaturation temperature of the target nucleic acid to release the labeling probe from the beads into the supernatant followed by measuring the amount of light or fluorescence in the supernatant over time (see, for example, Japanese Unexamined Patent Application, First Publication No. 2004-121231).
In addition, a method has been disclosed that consists of introducing a reactive functional group into a base that composes an oligonucleotide, and forming covalent bonds between other oligonucleotides and other molecules through this reactive functional group (crosslinking). For example, examples of technologies for crosslinking nucleic acid molecules by covalent bonding using a base derivative introduced with a reactive functional group include a method that uses 2-amino-6-vinylpurine (see, for example, Sasaki, S., Yakugaku Zasshi, 2002, Vol. 122, No. 12, pp. 1081-1093), and a method that uses a photoreactive base derivative in the form of 3-cyanovinylcarbazole nucleoside (see, for example, Fujimoto, et al., Nucleic Acids Symposium Series, 2008, Vol. 52, pp. 423-424; Yoshimura, et al., Organic Letters, 2008, Vol. 10, No. 15, pp. 3227-3230; International Publication No. WO 09/066,447).
Other methods consist of quantifying a nucleic acid molecule by using a photocrosslinking reaction and fluorescence resonance energy transfer (FRET). For example, a method has been disclosed that consists of associating a FRET probe and target nucleic acid molecule under conditions suitable for specific association followed by forming covalent bonds by using a photocrosslinking reaction between the two nucleic acid strands of the association product formed without changing the temperature or salt concentration of the reaction solution, and then detecting and analyzing this association product for each molecule thereof (see, for example, Japanese Unexamined Patent Application, First Publication No. 2011-036150). In the case of detecting using hybridization, although there is increased susceptibility to the formation of non-specific association products (association products formed by non-specific hybridization) during the association product detection procedure since detection of the association product formed is typically carried out under ordinary measurement temperature conditions (such as room temperature), in the case of the previously described method, the formation of non-specific association products can be effectively suppressed due to stabilization of the association product formed between the FRET probe and target nucleic acid molecule by a photocrosslinking reaction.
A FRET probe that specifically binds to a specific nucleic acid molecule is also used to detect antigen-antibody reactions. For example, a method has been disclosed that consists of carrying out an antigen-antibody reaction using an antibody labeled with single-stranded DNA and binding the antigen-antibody complex formed with a FRET probe complementary to the single-stranded DNA used to label the antibody, followed by releasing the fluorescent substance in the FRET probe from the antigen-antibody complex into the reaction liquid supernatant by treating with a nucleolytic enzyme, and detecting the antigen by measuring fluorescence intensity of the reaction liquid supernatant (see, for example, Japanese Unexamined Patent Application, First Publication No. 2011-033613).